Bacteria have been playing an important role in development of biotechnology. Especially, since the structure of DNA double helix was discovered by James Watson and Francis Crick, DNA manipulation techniques of microorganisms have been applied to produce a variety of useful materials, and thus, it has become possible to use microorganisms in wide area of industrial applications due to DNA manipulation techniques and techniques for efficiently producing various useful materials have been developed together with the DNA manipulation techniques. Recently, biotechnology, which started with simple protein production, has broadened its application area in various fields.
Among techniques newly introduced based on the remarkable development of biotechnology, there is a cell surface display technique based on a basic knowledge of Molecular biology and a technique of secretion and expression of proteins. Cell surface display is a technique in which proteins or peptides are fused with a proper cell surface anchoring motif to express on the surface of Gram-positive and Gram-negative bacteria, fungus, yeast and animal cells (Lee, S. Y. et al., Trends Biotechnol., 21:4552, 2003). The first cell surface display technique, in which peptides or small proteins are fused with pIII of filamentous phage and named surface-expression system, was developed by George P. Smith in the mid-1980s. Since then, a new way of cell surface display in which desired proteins are stably expressed on the surface of a microorganism was brought to the lime light, as many studies on secretion mechanism in microorganisms are being conducted.
In prior art, studies on the expression of foreign proteins on the surface of phage was conducted because the surface of phage is simpler than that of bacteria. Cell surface display using phages was used in the screening of antibodies, epitopes, high-affinity ligands, etc, but it has a disadvantage in that the size of a protein, which can be expressed on the surface of phage, is relatively limited (Georgiou, G. et al., Nat. Biotechnol., 15:29, 1997). Therefore, as one of the alternatives, cell surface display techniques were developed, in which target proteins are stably expressed on the surface of a microorganism, using surface proteins of microorganisms such as bacteria or yeasts.
Meanwhile, Gram-negative bacteria have a very unique membrane structure consisting of inner cell membrane, periplasmic space, and outer cell membrane. In order to express a foreign protein on the surface of a bacterium, such as E. coli having the above mentioned membrane structure, a cell surface anchoring motif, capable of stably and efficiently deliver the foreign protein to be expressed to the surface of a cell, is required. In order to express a foreign protein on the surface of a cell using surface proteins of bacteria, the foreign protein is fused with a proper surface protein at the genomic level to biosynthesize a fusion protein, and the obtained fusion protein should pass through an intracellular membrane to attach on the surface of a cell and maintained. For this, a surface protein having the following properties should be selected and used as a cell surface anchoring motif, the properties are as follows.
The surface protein has a very efficient secretion signal sequence helping a foreign protein to pass through an inner cell membrane in order to deliver it to the surface of a cell, and a targeting signal helping the foreign protein to be stably attached on an outer cell membrane surface, and at the same time, it is capable of delivering a large size of foreign proteins and stably expressing a large amount of the foreign proteins. Cell surface anchoring motifs used until now in E. coli were proteins present on the outer membrane such as LamB, PhoE (Agterberg, M. et al., Gene, 88:37, 1990), OmpA and so on.
However, when the above mentioned proteins are used, it is advantageous in that a foreign protein can be inserted into a loop protruding form the surface of a cell to be successfully expressed on the surface of a cell, but is has a disadvantage in that the size of the foreign protein which can be inserted, is limited with respect to its structure (Georgiou, G. et al., Nat. Biotechnol., 15:29, 1997).
Also, since the C-terminal end and the N-terminal end of the inserted foreign proteins need to be close three-dimensionally, when both ends are distanced, the both ends need to be genetically engineered to be located closely to one another using a binding peptide. In fact, in the case of LamB or PhoE, when a foreign protein having more than 50-60 amino acids was inserted, a stable membrane protein cannot be formed due to structural limitation (Stahl, S. et al., Trends Biotechnol, 15:185, 1997).
When a target protein is fused with a selected surface anchoring motif to express on the surface of a cell, until now, three general fusion methods have been used for the expression on the surface of a cell. The methods are as follows; A method in which a foreign protein to be expressed is fused with the C-terminal end of a surface anchoring motif or a foreign protein to be expressed is fused directly with a surface anchoring motif after deleting the C-terminal end of the surface anchoring motif (C-terminal deletion fusion). INP using levansurase for bioconversion (Jung et al., Nat. Biotechnol., 16:5142, 1999), an outer cell membrane protein C (OmpC) surface-expressing polyhistidine (Xu and Lee, Appl. Environ. Microbiol., 65:5142, 1999), FadL used in reactions exhibiting enantioselectivity of lipase ((Lee et al., Appl. Environ. Microbiol., 70:5074, 2004) and OprF (Lee et al., Appl. Environ. Microbiol., 71:8581, 2005) can be used in this method.
On the contrary to the above method, a method in which a target protein to be expressed is fused with the N-terminal end of a surface anchoring motif. Gram-positive bacteria such as Staphylococcus aureus protein A (Gunneriusson, E. et al., J. Bacteriol, 178:1341, 1996), fibronectin binding protein B of Staphylococcus aureus (Stasuss, A. et al., Mol. Microbiol., 21:491, 1996), fibrillar M protein of Staphylococcus pyogenes (Pozzi, G. et al., Infect. Immun., 60:1902, 1992) can be used in this method. Gram-negative bacteria, for instance, E. coli cannot be used in this method.
And, sandwich fusion method in which a foreign protein to be expressed on the surface of a cell is fused between the proteins used as a surface anchoring motif. Various proteins, such as PhoE (Agterverg, M. et al., Gene, 88:37, 1990), FimH (Pallesen, L., Microbiology, 141:2839, 1995), and PapA (Steidler, L. et al., J. Bacteriol., 175:7639, 1993) are used to express the target protein on the surface of E. coli. But this method could not overcome disadvantages that foreign proteins are not easily fused, expressed foreign proteins are frequently inactivated and the size of the foreign protein that can be inserted is limited to 60˜70 amino acids (Georgiou, G. et al., Nat. Biotechnol., 15:29, 1997; Stahl, S. et al., Trends Biotechnol., 15:185, 1997).
As described above, the application area of cell surface display employing bacterial secretion system in Biotechnology is wide, and it is determined by the type of a foreign protein to be expressed on the surface of a cell. A bacterial vaccine containing epitopes originated from a pathogen, which is expressed on cell surface of a cell, can be applied to patients to induce stronger and more sustained immune response than conventional vaccines using attenuated pathogenic microbes or viruses (Nguyen, T. N. et al., Gene, 128:89, 1993). The screening of various peptides or antibodies, when a specific Fab or a single chain antibody as well as the above mentioned recombinant live vaccines is expressed on the surface of a cell, can be simply performed (Francisco, J. A. R. et al., Proc. Natl. Acad. Sci. USA, 1:10444, 1993). In addition, this technique is highly valuable for application such as antibody production by administrating bacteria expressing a specific antigen to animals (Charbit, A. et al., Gene, 70:181, 1988), whole cell absorbents applying to heavy metal elimination or waste water treatment using microbes expressing metal absorbing proteins on the cell surface (Sousa, C. et al., J. Bacteriol., 180:2280, 1998), and whole-cell bioconversion utilizing enzymes used for biological conversion, which is stably expressed on the cell surface and used continuously without a decrease in catalytic activity (Richins, R. et al, Nat. Biotechnol., 15:984, 1997).
Therefore, there is an urgent need in the art to develop a cell surface display technique which is useful in the various field of biotechnology, especially the development of a method in which a target protein is stably and effectively expressed in large amounts on the surface of bacteria, a new surface anchoring motif and a surface expression vector using the same.
Accordingly, the present inventors have made extensive efforts to develop a surface anchoring motif, which can effectively express a foreign protein on the surface of a microorganism, deliver the expressed protein to the surface of the cell, and stably overexpress a foreign protein with large size, and a vector using the same, and as a result found, when a recombinant expression vector was constructed using exosporium protein BclA derived from Bacillus anthracis as a cell surface anchoring motif, a foreign protein is effectively overexpressed on the surface of a transformant, thereby completing the present invention.